Spider silk and spider webs have attracted the interest of man since ancient times [Lewis R. V. Chem. Rev. 2006, 106:3762-3774]. For several centuries, scientists have realized the potential of using spider silk as a material in the use of mankind, taking advantage of its remarkable strength. However, unlike silk, which is commercially produced from silkworms in quantities, spiders cannot be cultured in captivity due to their territorial and aggressive behavior. Thus, an artificial system must be used to synthesize spider silk.
Spiders have impressive fiber spinning abilities and produce up to seven different types of silk, each of which is stored and secreted by a specialized gland and utilized for a specific function during the life time of the spider [Gosline, J. M. et al., J. Exp. Biol. 1999, 202 Part 23, 3295-3303; 115; Lewis, R. V. Chem, Rev, 2006, 106, 3762-3774]. Among the different types of spider silk, the “dragline silk” is studied most intensely. The dragline silk is used by the orb-web weaving spiders to construct the frame and radii of their webs as well a life line when they fall or escape danger [Vollrath, F. and Knight, D. P. Nature 2001]. To be able to perform these tasks, the dragline fiber displays a remarkably high toughness due to combination of high elasticity and strength, which places it as the toughest fiber, whether natural or man-made [Gosline, J. M. et al., J. Exp. Biol. 1999, 202 Part 23, 3295-3303; Lewis, R. V. Chem, Rev, 2006, 106, 3762-3774]. For instance, dragline is six times as strong as high-tensile steel in its diameter and three times tougher than Kevlar that is one of the strongest synthetic fibers ever made [Gosline J. M. et al., J. Exp. Biol. 1999, 202 Pt 23:3295-3303]. Thus, it is no wonder that this material is regarded as having a huge potential as a biomaterial in the service of man [Lewis R. V. Chem. Rev. 2006, 106:3762-3774].
The building blocks of this extraordinary biological polymer are two paralogous structural proteins belonging to the fibroins family, secreted from the major ampullate gland, named MaSp1 and MaSp2 [Winkler, S. and Kaplan, D. L., J. Biotechnol. 2000, 74, 85-93].
These proteins were found to be composed of three different domains; a predominant central repetitive core consisting of hundreds of repeats, flanked with much shorter, nonrepetitive N and C-terminal domains [Ayoub, N. et al., PLoS One 2007, 2, e514], both non-repetitive domains are most highly conserved between different spider species. The C-terminal domain is important for the formation of the proper structure and assembly of the dragline fiber [Ittah, S. et al., Biomacromolecules 2006, 7, 1790-1795; Ittah, S. et al., Biomacromolecules 2007, 8, 2768-2773]. The N-terminal sequence, which contains a signal peptide, may also have a structural role but probably mainly serves to secrete the proteins into the lumen of the major ampullate gland [Ayoub, N. A. et al., PLoS One 2007, 2, e514; Hayashi, C. Y. and Lewis, R. V. Science 2000, 287, 1477-1479]. Only lately the full sequences of entire dragline spider silk proteins of the black widow were derived, which has unraveled the huge size of their genes (˜9.4-11.3 kb) and their precise genomic structure [Ayoub, N. A. et al., PLoS One 2007, 2, e514; Hayashi, C. Y. and Lewis, R. V. Science 2000, 287, 1477-1479].
The reiterated repeats sequences of these proteins vary slightly, but always contain poly-A stretches, separated by glycine-rich motifs such as GPGXX or GGX [Gosline, J. et al., J. Exp. Biol. 1999, 202 Part 23, 3295-3303; Lewis, R. V. Chem. Rev. 2006, 106, 3762-3774;]. Poly-A stretches are known to adopt a β-strand structure and a number of β-strands gather to create a β-sheet. The latter have the ability to create an ordered structure known as β-pleated sheet—a layered structure that is energetically preferred due to the burying of the hydrophobic methyl side chain of the alanines.
This structure gives rise to the mini-crystals found in native dragline silk, and due to its highly ordered structure, this domain is considered to be responsible for the typical melting point of the fiber [Cunniff, P. M. Polym. Adv. Technol. 1994, 5, 401-410]. The glycine-rich regions are sometimes referred to as “amorphous” and thought to adopt a 310 helix structure as well as β-turns and coils, loose structures that are considered to confer the final fiber its elasticity property [Hayashi, C. Y. et al., Int. J. Biol. Macromol. 1999, 24, 271-275].
Most araneoid spider species produce an MaSp1 fibroin, which does not contain prolines in its repetitive sequences and a proline-rich MaSp2 fibroin. However, A. diadematus secretes two proline-rich dragline fibroins, ADF3 and ADF4, which may thus be considered as MaSp2 proteins [Gatesy, J. et al., Science 2001, 291, 2603-2605]. Previously, the inventors have suggested that comparison of the sequences of these two fibroins to other MaSp fibroins reveals that ADF4, presented in FIG. 6, like all the MaSp1 fibroins that were analyzed, was more hydrophobic than ADF3 and the MaSp2 fibroins [Huemmerich, D. et al., Curr. Biol. 2004, 14, 2070-2074]. ADF4, like MaSp1 fibroins, also has a low content of glutamine, which is a polar amino-acid, in contrast to ADF3 and MaSp2 fibroins that have a higher glutamine content and display a QQ motif in their repeats. Thus, except for the proline content ADF4 resembles MaSp1 fibroins, and as described in the following, it tends to aggregate into fibers under several different experimental conditions, most likely due to its hydrophobic nature.
The scope of applications for which dragline spider silk may be employed is very wide and is only limited by the imagination of the beholder. It has been suggested for instance that if this material can be manufactured at a relatively large scale then it may be used for making low weight bullet-proof vests, new type of enforced fishing lined as well as a new type of textile. It has been suggested for use in biomedicine as biodegradable surgical sutures for microsurgery and in electronics as microconductors if the microfibers can be coated by heavy metals binding to amino acid analogues [Vollrath F. and Knight D. P. Nature 2001, 410:541-548]. However, since spiders cannot be cultured in captivity due to their solitary and predatory nature, in order to efficiently synthesize spider silk a heterologous artificial system must be used.
During the last two decades, many attempts were made to artificially synthesize spider silk proteins in heterologous hosts using genetic engineering techniques. Cloning of partial cDNAs coding for dragline proteins was achieved by several groups [Guerette, P. A. et al., Science 1996, 272, 112-115; Hinman, M. Results Probl. Cell. Differ. 1992, 19, 227-254; Rising, A. et al., Insect Mol. Biol. 2007, 16, 551-561]. Expression of natural and synthetic recombinant dragline silk proteins took place using bacteria, yeast, plants, mammalian cells, and transgenic goats [Lewis, R. V. Chem. Rev. 2006, 106, 3762-3774]. However, due to the highly repetitive nature of the sequence, as well as the restricted pool of amino acids composing the major part of the proteins, transcription and translation of the sequences were usually problematic, resulting in premature termination and low yields. Previous attempts to purify soluble dragline proteins, whether native or synthetic and artificially spin them, resulted in fibers with inferior properties as compared with the native dragline fiber [Brooks, A. E. et al., Biomacromolecules 2008, 9, 1506-1510].
Lately, a new approach to overcome the problematic solubility of spidroins led to successful self-assembly of dragline-based synthetic proteins [Stark, M. et al., Biomacromolecules 2007, 8, 1695-1701]. In this study, synthetic constructs containing four repeats of poly-A and glycine-rich regions, ending with the native C-terminal domain of MaSp1 originated from E. australis, were fused to a solubility enhancing fusion protein, thioredoxin. The protein was expressed in E. coli and remained soluble after its purification. Cleavage of the fusion protein initiated spontaneous self-assembly of the synthetic dragline proteins into fibers that were tested and found to be of high tensile strength and toughness but inferior to that of the native dragline fiber of this species [Stark, M. et al., Biomacromolecules 2007, 8, 1695-1701; Hedhammar, M. et al., Biochemistry 2008, 47, 3407-3417].